The complex interactions of the various cells involved in the generation of the in vivo human immune response can not be adequately reconstituted in vitro. Studies of the human immune system and the development and testing of immunomodulators suitable for human administration have been restricted by the limited ability to experiment with human subjects and the lack of animal models of the human immune system. For example, a major limitation to the preclinical evaluation of novel cancer immunotherapies is the lack of an animal model which contains a functional adult human immune system, having its hemopoietic and lymphopoietic tissues, particularly the spleen, reconstituted with the range of functional cells, including subtypes of T cells, such as T helper cells and cytotoxic cells, B cells and natural killer (NK) cells necessary to reconstitute the complex cellular interactions of the cellular and humoral human immune system.
Several methods have been developed to transfer the human immune system to mice. One approach for generating animal models has involved the use of immunocompromised hosts, which do not mount an effective immune response against the xenogenic hemopoietic cells of the human immune system. The immunocompromised hosts are engrafted with cellular elements of the human immune system.
Reconstitution has been attempted in genetically immunocompromised mice, such as severe combined immunodeficient (SCID) mice and Bg/Nu/XID mice, Homozygous C.B-17 scid/scid mice (SCID mice), congenic partners of BALB/Can, have a severe combined immunodeficiency and lack functional T and B cells (McCune, J. M. et al., Science 241:1632, 1988). SCID mice are unable to mount an effective cellular or humoral response to foreign antigens. Reconstitution has also been attempted in mice in which an immunocompromised state has been induced by for example radiation (Abedi, M. R. et al, Eur. J. Immunol. 22:823, 1992), and cytotoxic drugs (Huppes, W. et al, Eur. J. Immunol. 22:197, 1992).
The strategies that have been used for preparing mice reconstituted with the human immune system involve the transplantation of human fetal tissue (McCune et al., Science 241:1632, 1988), injection of human peripheral blood lymphocytes (PBLs) (Mosier et al., Nature 335:256, 1988), and transfer of bone marrow (Kamel-Reid & Dick, 1988; Lubin et al 1991) into immunodeficient mice such as SCID and beige nude mice.
More particularly, McCune et al (Canadian Patent Application No. 2,048,406 and Science 241: 1632, 1988) engrafted SCID mice with solid implants of fetal human tissue, such as lymphoid tissue, thymus, spleen, bone marrow and hematopoietic stem cells. Fragments of tissue or cells from fetal liver and thymus were introduced into SCID mice. Human lymphocytes were detected in recipient mice, but human cells did not reconstitute host lymphopoietic tissue and were not detected in the host thymus or spleen.
McCune et al also pre-treated the SCID mice with either fractionated courses of irradiation or an anti-asialoglycoprotein antibody (anti-ASGM1), a rabbit antiserum against murine NK cells and macrophages, to at least partially ablate endogenous hematopoietic cells prior to implant of the xenogenic tissue. However, human T cells were only transiently detected in the host peripheral blood and did not appear to home to the host spleen, thymus or lymph nodes. Human IgG production required the introduction of human lymph nodes into the SCID mice.
SCID mice have also been engrafted with post-natal thymus tissue following pretreatment with either radiation or administration of anti-ASGM1, a rabbit antiserum against murine NK cells and macrophages (Barry, T. S. et al, J. Exp. Med. 173:167, 1991). Treatment with anti-ASGM1 promoted thymus engraftment, T and B lymphopoiesis and production of mouse immunoglobulin in the SCID mice. However, only rare CD4+ and CD8+ cells were detected in the spleen.
Mayo et al (PCT Application No. WO91/16910) transplanted SCID mice with human lymph nodes and fetal long bone but did not identify homing of human lymphocytes to the spleen.
An alternative technique for reconstituting mice, developed by Mosier et al, does not require the procurement of human fetal tissues (Mosier et al, Nature 335:256, 1988 and PCT No. WO 89/12823). The Mosier protocol involves injecting adult peripheral blood leukocytes (PBL) into the peritoneal cavity of SCID mice to yield mice referred to as human-peripheral blood leukocytes-SCID (Hu-PBL-SCID) mice.
There are several limitations to the Hu-PBL-SCID model, as originally described by Mosier et al. Initially Mosier et al reported a high level of human lymphocytes in the host spleen. However, this high level of splenic engraftment was subsequently attributed to non-specific binding of the antibodies used to differentiate the human cells from mice splenic cells (Mosier, Nature, 338:211, 1989). Others have also been unable to achieve high levels of splenic reconstitution following the method of Mosier et al when rigorous controls were employed to eliminate non-specific antibody staining (Carlsson et al, J. Immunol. 148:1065, 1992). Jicha, D. L. (J. Immunol. 11:19, 1992), also failed to detect human cells in the spleens of Hu-PBL-SCID mice prepared by the Mosier protocol.
Following the Mosier protocol, the level of human PBLs engrafted in the mice is often low and quite variable. Human T cell engraftment in the lymphoid organs of the Hu-PBL-SCID mice is also significantly limited during the early post-reconstitution period as human PBLs appear to remain predominantly in the peritoneal cavity during the first weeks after reconstitution (Mosier et al., Nature 335:256, 1988; Tary-Lehmann and Saxon, J. Exp. Med. 175:503, 1992; Pfeffer et al., Curr. Top. Microbiol. Immunol. 152:211, 1989; Krams et al, J. Exp. Med. 170:1919, 1989; Torbett et al., Immunol. Rev. 125:139, 1991; Mosier et al., Nature 388:211, 1989; Duchosal et al., Am. J. Path. 141:1097, 1992). Consequently, it has been difficult to assess the functional activities of the engrafted human cells. Human natural killer (NK) activity in the Hu-PBL-SCID model has also not been demonstrated. Hu-PBL-SCID mice produced by the Mosier protocol do not exhibit significant graft versus host reactions and may live for several years after transplant (Duchosal, Cellular Immunol. 139:468, 1992). Accordingly, the mice produced using the Mosier Hu-PBL-SCID protocol are not useful models for studies of human graft versus host disease.
Not all human immune functions can be induced in the Mosier Hu-PBL-SCID mouse model. For example, while it is possible to induce human secondary immune response to tetanus toxoid, and hepatitis B core antigens (Hbc) (Mosier, PCT No. WO89/12823; Duchosal PCT No. WO 93/05796; Duchosal et al Am. J. Pathol. 141:1097, 1992; Duchosal et al, Am. J. Pathol. 141:1097, 1992), no studies have been published, that clearly demonstrate a human primary immune response in this system. The demonstration of a human primary immune response in a non-human host may be a key factor in demonstrating functionality of the xenogenic immune system in the host.
The induction of a human primary immune response in SCID mice engrafted with human PBLs using the Mosier protocol has been the subject of many investigations but no results have been published that clearly and consistently demonstrate this characteristic function in the mice. For example, Mazingue et. al., (Eur. J. Immunol. 21:1763, 1991) reported a primary immune response to a schistosome antigen in Hu-PBL-SCID mice. However, no data for IgM response specific for this antigen was presented, only two Hu-PBL donors were tested, and a single antigen was tested for primary immune response in this model. Mazingue et al also expressed doubts on the human nature of antibodies detected in the Hu-PBL-SCID mouse serum as the anti-human IgG reagents used reacted with mouse sera also. Markham and Donnenberg (Inf. & Immun. 60:2305, 1992) tried to evoke human primary immune response against keyhole limpet haemocyanin (KLH) in the Hu-PBL-SCID mice, but were unsuccessful.
Abedi et. al. (Eur. J. Immunol. 22:823, 1992) tried to induce human primary immune response in Hu-PBL-SCID mice to hepatitis B surface antigen. However, only two out of the sixteen mice were reported to give IgG and IgM response, and no antibody titre results were published for these animals. The authors of this study subsequently noted that the majority of the mice had failed to respond and that they could not exclude a fortuitous expansion of hepatitis-B specific clones in selected mice (Smith, C. I. E. et al, Immunol. Rev. 124:113, 1991).
Duchosal et. al. (Nature 355:258, 1992) engrafted SCID mice with human PBL from a donor with no detectable anti-Hbc antibody titre. One out of twelve Hu-PBL-SCID mice were reported to produce a weak transient IgM and no IgG response after Hbc immunization. Duchosal et al described the ability of Hu-PBL-SCID mice to mount a low primary response to tetanus toxoid (Duchosal, M. A. et al, PCT No. WO93/05796). Although the donors in that study had not been exposed to the antigen recently, it was not known if the donors had some prior exposure to the antigen. The lack of consistent human primary immune response reported in the studies using the Mosier et. al. (Nature, 335:256, 1988) engraftment procedure may be attributed to the low levels of human lymphocyte engraftment.
Evidence of a primary immune response has also been reported from chimeric mice with double engraftments of human bone marrow and from SCID mice transplanted with human lymph nodes. Reisner (Reisner Y., European Patent Application No. 438,053) reconstituted lethally irradiated BALB/c mice with human bone marrow cells and supportive bone marrow cells from SCID mice and reported a primary immune response to KLH-DNP in two mice. Mayo et al (Mayo, S. et al., PCT No. WO91/16910) transplanted human lymph nodes into SCID mice and immunized the graft with TNP-KLH. Four days later anti-TNP producing cells in the lymph node graft were identified by immunohistology. Human antibody producing cells were not shown to have reconstituted host lymphopoietic tissue.
Functional human T, B and NK cells have not been demonstrated in the Mosier Hu-PBL-SCID model. Only limited engraftment of human lymphocytes in the spleen has been achieved with the Mosier protocol discussed above. It is noted that SCID mice posses high levels of active NK cells (Dorshkind et al., J. Immunol. 134:3798, 1985) which can represent a barrier to lymphoid allografts (Sheng-Tanner and Miller, J. Exp. Med. 176:407, 1992) as shown recently (Murphy et al., Eur. J. Immunol. 22:1421, 1992). Pre-treatment with radiation or anti-ASGM1 prior to transplant has been reported to increase engraftment of human lymphocytes, although the reports have provided conflicting results. Low doses of total body irradiation of SCID mice (up to 4 Gyg) have been reported to have minimal effect on engraftment or development of graft versus host disease (GVHD), whereas proliferation of human cells was reported to increase by treatment with anti-mouse IgM (Huppes, W. et al, Eur. J. Immunol. 22:197-206, 1992). Prior irradiation of SCID mice has been reported to increase human IgG levels in the first month after transplant of the Hu-PBL (Duchosal, Am. J. Pathol. 141:3097, 1992). Prior irradiation may cause a slightly more rapid appearance of human IgG in the serum, but does not appear to potentiate repopulation of human cells in the mouse (Smith, C. I. et al, Immunol. Rev. 124:113, 1991). Pre-treatment of mice with anti-ASGM1 has been reported to increase the number of human cells in the spleen one month after engraftment (Murphy, W. J. et al Eur. J. Immunol. 22:1421, 1992). However, the results were variable with 8 out of 28 mice having no detectable human cells in the spleen and the remainder having levels ranging from 1 to 48%.
Human lymphocytes have been detected in the spleen of Hu-PBL-SCID mice by reaction with the human leukocyte common antigen CD45 (Huppes, W. et al, Eur. J. Immunol. 22:197, 1992). In particular, the presence of human T cells has been detected using the CD3, CD4 and CD8 markers (Huppes, 1992, supra; Davies, Clin. Immunol. Immunopath. 60:319, 1991; Murphy, Proc. Nat. Acad. Sci. USA 89:4481, 1992). Human NK cells (CD16+) have only been detected in the peritoneal cavity of the Hu-PBL-SCID mice, the site of injection of the Hu-PBL, and functionality in lysing specific targets has not been determined for these NK cells (Torbett, B. E. et al, Immunol. Rev. 124:,139, 1991).
Animal models of the human immune system are expected to be particularly useful for the evaluation of the effect of drugs on the human immune system, for the development of therapeutics and immunizing compositions and vaccines. Such models would also be useful for testing methods of preventing, treating, enhancing or suppressing an immune response and for treating diseases, such as cancer or infection by pathogenic organisms.
SCID mice reconstituted using lymphocytes have been used to study infectious diseases (McCune et al, 1990, Cannon et al 1190, McCune et al 1991, Okano et al, 1990, Mazingue et al, Eur. J. Immunol. 21:1763, 1991) tumors (Kamel-Reid et al, Science, 246:1597, 1989; Charley et al, J. Invest. Dermatol. 94:381, 1990) autoimmunity (Krams et al, J. Exp. Med. 170:1919, 1989; Duchosal et al, J. Exp. Med. 172:985, 1990; Macht et al., Clin. Exp. Immunol. 84:34, 1991) and primary immunodeficiency (Smith et al, Immunol. Rev. 125:113, 1990 and; Saxon et al, J. Clin. Invest. 87:658, 1991).